The contribution of inflammatory mechanisms to the pathogenesis of transplant rejection is the major focus of this program project. Many key inflammatory mediators act through G protein-coupled receptors (GPCRs) that transduce ligand-dependent signals via hetero-trimeric guanine nucleotide-binding proteins (G proteins). During the previous grant period, we showed that two GPCRs, the AT1 receptor for angiotensin II and the TP receptor for thromboxane A2, promote alloimmunity and graft rejection. Based on the similar actions of these Gq-coupled receptors, we hypothesize that allograft rejection is enhanced through distinct GPCR signaling pathways coupled to Gq proteins and to the activation of Rho GTPases and Rho-kinase. Moreover, we suggest that these pathways influence graft function through discrete actions upon the T cell, upon the macrophage/antigen-presenting cell, and upon the allograft itself. We will test this hypothesis using genetic models with inducible activation or inhibition of Gq and Rho-kinase. By modulating Gq and Rho-kinase signaling in models of transplantation generated through the program core, the contributions of these pathways to the pathogenesis of rejection will be precisely defined. Our proposed studies have the following specific aims: 1. To define the capacity of Gq signaling to promote cellular immune responses. 2. To characterize the actions of Gq signaling in organ transplant rejection. 3. To define the interactions between G proteins and Rho-GTPases in immune cells. 4. To identify the cellular mechanisms of action of Rho-kinase in rejection. Because the large family of GPCRs uses only a limited number of G proteins, we posit that G protein signaling pathways that enhance inflammation and graft injury may be useful therapeutic targets whereby the actions of multiple pro-inflammatory ligands could be blocked by targeting a single common pathway.